Comparative proteomic profiling of the ovine and human PBMC inflammatory response

Understanding the cellular and molecular mechanisms of inflammation requires robust animal models. Sheep are commonly used in immune-related studies, yet the validity of sheep as animal models for immune and inflammatory diseases remains to be established. This cross-species comparative study analyzed the in vitro inflammatory response of ovine (oPBMCs) and human PBMCs (hPBMCs) using mass spectrometry, profiling the proteome of the secretome and whole cell lysate. Of the entire cell lysate proteome (oPBMCs: 4217, hPBMCs: 4574 proteins) 47.8% and in the secretome proteome (oPBMCs: 1913, hPBMCs: 1375 proteins) 32.8% were orthologous between species, among them 32 orthologous CD antigens, indicating the presence of six immune cell subsets. Following inflammatory stimulation, 71 proteins in oPBMCs and 176 in hPBMCs showed differential abundance, with only 7 overlapping. Network and Gene Ontology analyses identified 16 shared inflammatory-related terms and 17 canonical pathways with similar activation/inhibition patterns in both species, demonstrating significant conservation in specific immune and inflammatory responses. However, ovine PMBCs also contained a unique WC1+γδ T-cell subset, not detected in hPBMCs. Furthermore, differences in the activation/inhibition trends of seven canonical pathways and the sets of DAPs between sheep and humans, emphasize the need to consider interspecies differences in translational studies and inflammation research.


Sample collection and ethics approval
This study was carried out using peripheral blood obtained by venipuncture from the jugular vein of six healthy adult, 3-4-year-old Merino ewes, with ethical approval by the institutional ethics and animal welfare committee and the national authority (license BMWF-68.205/0116-V/3b/2018).All methods were performed in accordance with the relevant guidelines and regulations implemented at the University of Veterinary Medicine Vienna, the Institutional Ethics Committee ("Ethics and Animal Welfare Committee") of the University of Veterinary Medicine Vienna.
All sheep included in the study were in a similar reproductive period (nongravid seasonal polyestrous) to ensure consistency in physiological conditions.They were confirmed to be systemically healthy by physical examination and the absence of hematologic abnormalities on complete blood count (CBC).Samples for CBC were subjected to routine blood cytometry performed by the University´s certified diagnostic laboratory within
To determine the optimal ratio for blood dilution, anti-coagulated whole blood (n = 3 donors) was used either undiluted or diluted in a 1:1 or 1:2 ratio with complete RPMI1640 medium (Gibco, Life Technologies, Austria).This medium was supplemented with 10% heat-inactivated fetal calf serum (Gibco, Life Technologies, Austria), and 1% Penicillin, Streptomycin, and Amphotericin (Sigma-Aldrich, Germany,complete medium).The processed blood samples were then layered over three different density gradient media: Ficoll-Paque PREMIUM® (1.077 g/ml gradient, Cytiva, Sweden), Percoll® (1.130 g/ml gradient, GE Healthcare Bioscience, Sweden), and Lymphoprep® (1.077 g/ml, STEMCELL Technologies, Germany).These samples underwent centrifugation at three different centrifugation forces: 300×g, 660×g, and 800×g, each for a duration of 30 or 60 min (min), at 21 °C, and without brakes.The result of these experiments was ranked according to the quality of separation and perturbation of the different layers (Erythrocyte/Granulocyte layer, density gradient medium, PBMC layer, Plasma layer) (Fig. 1b).
Subsequently, the two density gradient media (Ficoll 1.077 g/ml versus Lymphoprep 1.077 g/ml) and centrifugation times (660×g/30 min versus 660×g/60 min), that achieved the best separation quality, were selected for further optimization, aiming to identify the protocol yielding the highest PBMC count with minimal granulocyte contamination.To this end, PBMCs were collected from the medium-plasma interface using a sterile pipet into a 50 ml conical tube and washed once at 540×g for 10 min at 21 °C using 20 ml of PBS without calcium and magnesium (PBS−/−).Then, 5 ml erythrocyte lysis buffer, composed of 154 mM ammonium chloride, 10 mM potassium hydrogencarbonate, and 0.1 mM Ethylenediaminetetraacetic acid), was added to the cell pellet.The tube was gently shaken to facilitate dissolution of the pellet, incubated for 5 min on ice, then mixed with 15 mL of PBS−/−, and centrifuged at 450×g for 5 min at 21 °C.Finally, the supernatant was discarded, and the cell pellet was washed twice using 10 ml of washing solution (PBS−/− with 2% FCS) and centrifugation at 440×g for 5 min at 21 °C.After the final wash and removal of the supernatant, the PBMCs pellet was processed for further analysis.
The yield of PBMCs was quantified by counting live cells per unit volume, determined by microscopic enumeration using a Neubauer hemocytometer.Cell viability was defined as the proportion of live cells in a population, assessed by their ability to exclude Trypan blue dye.The composition and purity of PBMCs were quantified using the ADVIA 2120i Hematology System™ Automated Cell Counter (Siemens, Germany).The composition of PBMCs was determined by calculating the ratio of isolated lymphocytes and monocytes to the total number of isolated PBMCs population (no monocytes/no PBMCs and no lymphocyte /no PBMCs), with the results expressed as percentage.The purity was determined as the percentage of PBMCs in the total isolated leukocyte population, with a specific emphasis on assessing contamination with other cell types such as granulocytes and erythrocytes.To ensure the suitability of the isolated PBMCs for downstream applications, stringent criteria were set, demanding a minimum viability of 95% and a purity exceeding 95% 105,106 .

Proteomic phenotypic characterization of isolated oPBMCs
Due to the limited availability of ovine-specific antibodies essential for immunophenotyping techniques such as flow cytometry analyses 63,73 , oPBMCs were phenotypically characterized using MS-based proteomic analyses of lineage specific surface markers.The composition of PBMCs was determined based on the expression of cell type markers, while their purity was determined based on the presence or absence of granulocyte-specific CD antigens and specific proteins associated with platelets and plasma.
The PBMCs pellets were resuspended in serum-free medium (RPMI 1640 medium supplemented with 1% Penicillin, Streptomycin, and Amphotericin (Sigma-Aldrich, Germany) at a concentration of 4 × 10 6 cells/ml.The cell suspension was plated into a T-25 flask (Greiner Bio-One, Kremsmünster, Austria) at a seeding density of 0.6 × 10 6 cells per flask and incubated for 3 h at 37 °C in a humidified 5% CO2 incubator.
After the incubation time of the PBMCs, the conditioned medium was harvested into a 15 ml falcon tube, leaving approximately 1 ml medium in the culture flask.The adherent cells remaining in the flask were then gently detached using a cell scraper and combined with the previously transferred conditioned medium in the falcon tube.The cell suspension was centrifuged at 540×g for 5 min at 4 °C to pellet the cells and separate the supernatant.The supernatant was transferred into a new 15 ml falcon tube, centrifuged at 2000×g for 10 min at 4 °C and filtered through a 0.2 μm filter to remove potential remaining cells and cell debris.The filtered secretome was precipitated on ice cold 99.6% ethanol and stored at − 20 °C until further processing for isolation of secreted proteins.
The cell pellet obtained from the initial centrifugation was washed twice with 5 ml PBS−/− and centrifugation at 540×g for 5 min at 4 °C.Following the removal of the final wash solution, 200 μl of Sodium deoxycholate lysis bufferer (SDC) (4% sodium deoxycholate, 100 mM Tris HCl pH 8.5) was added to the cell pellet.The mixture was then heated at 95 °C in the water bath for 5 min to ensure complete lysis of the cells.The lysate was subsequently stored at − 80 °C until further proteomic processing for isolation cell lysate proteins.
The secretome and cell lysates obtained from unwashed PBMCs were used as control samples for the evaluation of cell purity.These cells were directly plated after the RBC lysis step as donor-matched control for each PBMC sample, bypassing the final two washing cycles, and were then designated for subsequent culture and analysis via mass spectrometry to measure cell type specific CD markers and specific proteins associated with platelets and plasma.

Inflammatory stimulation oPBMCs
For a standardized assessment of inflammatory responses between ovine and human PBMCs, we adopted an inflammation induction protocol in oPBMCs consistent with the approach used for hPBMCs we previously described 107 .
In brief, isolated oPBMCs (n = 3 biological replicates (3 donors), 3 technical replicates/donor/experimental group) were resuspended to a final concentration of 4 × 10 6 cells/ml in the complete RPMI 1640 medium supplemented with 1 μg/ml of lipopolysaccharide (LPS, Sigma-Aldrich, Merck, Darmstadt, Germany) in combination with 5 µg/ml of Phytohaemagglutinin (PHA, Sigma-Aldrich).The cell suspension was then seeded into a T-25 flask (Greiner Bio-One, Kremsmünster, Austria) at a density of 0.6 × 10 6 cells per flask and incubated at 37 °C in 5% CO2 for 6 h.PBMCs cultured in complete RPMI 1640 medium without LPS or PHA served as healthy control samples.Following the 6-h period of inflammatory stimulation, the culture medium was changed to serum-free RPMI medium and further incubated at 37 °C in 5% CO2 for 3 h.Finally, both the secretome and cells were harvested for mass spectrometry analyses, as detailed previously in "Proteomic phenotypic characterization of isolated oPBMCs" section.

Shotgun proteomics by LC-MS/MS
A quantitative LC-MS/MS of both the oPBMCs cell lysate and secretome of the washed versus unwashed PBMCs, as well as stimulated versus untreated PBMCs in sheep, was carried out.

Sample preparation
Proteomic samples were prepared using a modified version of a previously described protocol 108 and employing an adapted version of the EasyPhos platform 109 .PBMC cell pellets were thawed, and further lysed using the S220 Focused-ultrasonicator (Covaris, LLC., Woburn, MA, USA).The precipitated secretome proteins were centrifuged at 5000×g for 30 min at 4 °C and the resulting protein pellet was solubilized in SDC buffer.Protein concentrations were determined via bicinchoninic acid assay (BCA)-assay.Protein (20 µg/sample) was reduced and alkylated with tris(2-carboxyethyl) phosphine (TCEP) and 2-chloroacetamide (2-CAM) for 5 min at 45

Differentially abundant proteins
To compare the inflammatory responses and pathways between ovine and human PBMCs, proteomics data from oPBMCs were juxtaposed with that of hPBMCs, with both sets inflamed and analyzed through the same methodological approach 107 .The mass spectrometry proteomics data of hPBMCs were retrieved from the Pro-teomeXchange Consortium through the proteomics identification database (PRIDE) repository with the dataset identifier PXD001415 (https:// doi.org/ 10. 6019/ PXD00 1415).A two-sided Student's t test was performed to examine differences between the control group and activated group, and the difference in abundance level between the two groups was calculated.Proteins satisfying a false discovery rate (FDR) ≤ 0.05 (used as the threshold of the q-value) and fold change (FC) │ ≥ 2│were considered to be significantly different (differentially abundant proteins, DAPs).

Enrichment analysis
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the DAPs were performed using the Search Tool for Retrieval of Interacting Genes/proteins (STRING) database (version 11.5, https:// string-db.org/) [111][112][113][114][115][116] with a cut-off p < 0.05.The DAPs were assigned to their corresponding Gene Ontology branches (Biological Process, Molecular Function, and Cellular Component) and KEGG pathways, employing a species-specific background dataset for accurate comparison.Interactions analyzed were strictly confined to those substantiated by experimental evidence.

Protein-protein-interaction network construction and module analysis
Protein-protein interaction (PPI) networks were constructed using STRING (version 11.5; https:// string-db.org), applying active interaction sources supported by experiments and an interaction score ≥ 0.4 117 , to identify functional interactions of DAPs.The PPI Networks were visualized and analyzed using the Cytoscape software (version 3.9.1,www.cytos cape.org) and its Molecular Complex Detection (MCODE) and CytoHubba plugins [118][119][120] .MCODE was employed to identify the main clusters in the PPI networks applying a degree cutoff = 2, node score cutoff = 0.2, K-core = 2, max.depth = 100, and haircut cluster finding setting as visualization criteria 119 .Clusters with a score ≥ 5 were considered significant subnetworks.With these clusters as input, we used STRING again to construct the second PPI network for further comprehensive enrichment analysis.
CytoHubba was utilized to calculate and rank the node scores of DAPs within PPI networks based on three hub protein-based identification algorithms, including the degree of connectivity, Maximal Clique Centrality (MCC), and Maximum Neighborhood Component (MNC).The top 30 hub proteins identified by each algorithm were plotted using Venn diagrams to determine overlapping proteins.The top overlapping proteins within the main cluster were designated as hub proteins [120][121][122][123] .

Pathway analysis
Pathway analysis was executed for the whole data set between the compared groups using Ingenuity Pathway Analysis (IPA) (QIAGEN Bioinformatics) 124 .The proteomic data sets, which comprised UniProt identifiers, p-values, and fold changes of total identified proteins, were imported into Ingenuity Pathway Analysis (IPA) for core analysis.The core analysis was conducted with the setting of direct and indirect relationships between molecules based on experimentally observed data, considering data sources in human databases within the Ingenuity Knowledge Base.IPA predicted potential canonical pathways of the proteins in this study, which were classified as activated or inhibited based on the Z-score, a statistical result of differential protein expression based on fold changes.Visualizing differentially affected pathways under different conditions was completed using the comparison analysis feature in IPA and hierarchical clustering.Pathways with Z score ≥ 2.0 (absolute) and p < 0.05 in at least one of the conditions were considered significant and reserved for comparison.Terms were filtered with respect to functional plausibility.

Statistical analysis
Statistical analyses were conducted using GraphPad Prism software (version 8.4.3).Continuous variables were expressed as mean ± standard deviation (SD), and categorical variables were expressed as percentages.Before statistical analysis, we assessed the normality of the data using the Shapiro-Wilk test 125 .Since the p-values were greater than 0.05 (p ≥ 0.05), suggesting normal distribution of the data, we employed parametric tests for the analyses.The differences in PBMC yield, purity and composition between the various PBMC isolation protocols and in CD marker expression and ratio between control and activated hPBMCs and oPBMCs were analyzed using ANOVA with Sidak correction for multiple comparisons when applicable.A two-sided Student's t test was performed to analyze differences between the unwashed PBMCs and washed PBMCs.A p-value < 0.05 was considered significant.

Ethics approval
This study was carried out using peripheral blood obtained by venipuncture from the jugular vein of six healthy adult, 3-4-year-old ewes, with ethical approval by the institutional ethics and animal welfare committee and the national authority (license BMWF-68.205/0116-V/3b/2018) and in accordance with the ARRIVE guidelines.No human participants were involved in this study; human proteomics data were retrieved from the ProteomeXchange Consortium through the proteomics identification database (PRIDE) 167 repository with the dataset identifier PXD001415 (https:// doi.org/ 10. 6019/ PXD00 1415).

Ovine PBMCs isolation
The oPBMCs isolation protocol was optimized regarding key variables including blood dilution, density gradient medium, and centrifugation parameters such as force and duration.Isolation protocols with blood diluted at 1:1 or 1:2 ratios in complete RPMI medium, utilizing either Lymphoprep or Ficoll for density gradient separation, and centrifuging at 660×g for durations of 30 or 60 min, achieved optimal layer separation.These protocols delineated four distinct layers-erythrocyte/granulocyte, density gradient medium, PBMCs, and plasma-more effectively and without perturbation than methods using undiluted blood, Percoll as the separation medium, and centrifugation forces of 800×g or 330×g (Fig. 1b).
Prioritizing first the purity and then the yield of the PBMCs, the isolation technique using blood diluted 1:1 with complete RPMI medium, Lymphoprep density gradient, centrifugation at 660×g for 30 min and erythrolysis followed by two washing steps, proved most effective and was thus used for all subsequent experiments.
Analysis of the various immune cell surface marker ratios, including T-cells: monocytes (CD3:CD16, CD3:CD163), T-cells:B-cells (CD3:CD19), T-cells:natural killer cells (CD3:CD352), T-cells:dendritic cells (CD3:CD11c), and T-helper cells:T-cytotoxic cells (CD4:CD8) across ovine and human, in both healthy control and inflamed PBMCs, based on their specific CD marker expressions, revealed no significant effects of species or activation status on the overall ratio of immune cell surface markers (p > 0.05).However, differential trends in specific immune cell surface marker ratios under different conditions were observed.Notably, healthy oPBMCs exhibited a 2.09 to 3.53-fold increase in the ratio of T-cell surface markers to the other PBMC surface markers compared to healthy hPBMCs, except for CD3:CD352, which was higher in humans (Table 3, Suppl.Table 2).In inflamed PBMCs, ovine CD-marker ratios for T-cells: monocytes and T-helper-cells: T-cytotoxic-cells were 2.5 to 21.5-fold higher compared to hPBMCs, respectively, while the other ratios were similar across species.(Table 3, Suppl.Table 2).Comparing the control and activated groups of PBMCs, both oPBMCs and hPBMCs showed similar patterns of either increased (CD3:CD16, CD3:CD163) or decreased (CD3:CD19, CD3:CD352) ratios.However, CD4:CD8 increased and CD3:CD11c decreased in sheep, while remaining constant in humans (Table 3, Suppl.Table 2).

Mass spectrometry (MS)-based profiling
Upon inflammatory activation of PBMCs, the MS-based proteomic analyses profiled 4217 proteins in the whole cell lysates of oPBMCs and 4574 in hPBMCs, alongside 1913 proteins in the secretome of oPBMCs and 1375 in hPBMCs.This profiling was conducted after applying stringent filters for high confidence (FDR < 0.01 at both peptide and protein levels) and reproducibility, ensuring each protein was positively identified in at least 70% of the samples from one sample group.The comparative proteomic profiling of hPBMCs and oPBMCs demonstrated a notable interspecies overlap.Specifically, 47.8% of the proteins identified in the cell lysate (equivalent to 2790 proteins) and 32.8% of the secretome (comprising 988 proteins) were shared across both species.
Table 1.Comparative analysis of oPBMC isolation efficiency: impact of centrifugation duration and density gradient media on the cell number (mean ± s.d.) isolated per ml blood and the percentage of isolated PBMCs.www.nature.com/scientificreports/Venn analysis, capturing the overlap between 176 and 71 DAPs in human and sheep PBMCs, identified 7 overlapped DAPs (IL1B, IFIH1, CCL4, ISG20, IL1RN, APOBEC3A, and PDCD11), which were simultaneously associated with human and sheep activated PBMCs, as well as 169 human-specific DAPs (107 upregulated and 62 downregulated), and 64 sheep-specific DAPs (52 upregulated and 12 downregulated) (Fig. 2).The top 10 DAPs of activated PBMCs in humans and sheep are listed in Table 4.

Enrichment analyses
DAPs of activated PBMCs were significantly enriched in 68 GO terms in sheep and 310 GO terms in humans (FDR < 0.05), of which 16 were shared between ovine and human PBMCs, 52 were ovine-specific and 294 humanspecific (Suppl.Tables 5-7).
The shared biological process ontologies of DAPs included defense response, response to stress, immune response, defense response to virus, defense response to other organism, Inflammatory response, cellular response, interspecies interaction between organisms, response to other organism, innate immune response, and immune effector process.Molecular function ontology of DAPs was associated with protein binding, RNA helicase activity, and binding (Suppl.Table 7).
The first and the second clusters in sheep were associated with RNA metabolism, and regulation of translational initiation (Suppl.Table 9).The third cluster in sheep, including six DAPs, and the primary cluster in human, including 34 DAPs, were predominantly associated with inflammatory responses.In the main cluster of DAPs 45 GO terms related to inflammatory biological processes were significantly enriched (FDR < 0.05) in sheep and 324 GO terms in humans (Suppl.Tables 9, 10).Within this set, 29 biological process terms were shared between the main clusters of sheep and human PBMCs (Suppl.Table 11), while 16 were specific to sheep and 295 were specific to humans.The top 15 biological process terms within the main cluster of PBMCs in humans and sheep are shown in Fig. 3c,d.Venn analysis, capturing the overlap between three CytoHubba algorithms, identified 27 overlapped proteins for sheep and 28 for humans.Subsequently, the top overlapped proteins within the main cluster were designated as hub proteins.Within the main ovine cluster, the hub proteins were STAT1, IL1B, IRF4, STAT3, and IL17A, while in the main human cluster, they were CXCL10, CXCL8, IL1B, IL6, and TNF (Suppl.Tables 12-14).Remarkably, these hub proteins were identified as species-specific, with IL1B being the sole hub protein shared between activated PBMCs during the 6-h time course in both humans and sheep.Enrichment analysis confirmed the relevance of these hub proteins to inflammatory responses in both species.

Pathway analyses
Ingenuity pathway analysis of the differential proteomic expression profiles of hPBMCs secretome, oPBMCs secretome, hPBMCs whole cell lysate, and oPBMCs whole cell lysate, identified 17 canonical pathways that exhibited conserved activation/inhibition patterns across both species.Additionally, seven canonical pathways demonstrated divergent activation/inhibition patterns between both species (Table 5).
The top 5 cross-species conserved pathways that were activated in the secretome and whole cell lysate, were interferon signaling, inflammasome pathway, Pathogen Induced Cytokine Storm Signaling Pathway, NOD1/2 Signaling Pathway, and acute phase response signaling (Table 5).The top 5 cross-species pathways that were activated in the secretome and then inactivated at the level of the whole cell lysate in both species, were phagosome formation, CXCR4 signaling, IL-8 signaling, NF-κB Activation by viruses, and ERK/MAPK Signaling (Table 5).
The top 5 pathways with a species-specific activation pattern, that were activated in the secretome of both species but inactivated at the level of the whole cell lysate only in sheep, were IL-6 signaling, IL-17 signaling, p38 MAPK signaling , HMGB1 Signaling, and S100 Family Signaling Pathway (Table 5).

Discussion
Sheep are commonly employed as a large animal model in immune-related studies 62,66,67,70 .However, inherent differences between human and sheep PBMCs may impact the translational relevance of research findings derived from sheep models.Therefore, in this cross-species comparative study, we examined the similarities and differences of the in vitro inflammatory response of ovine and human PBMCs by employing mass spectrometry to analyze the proteome of the PBMCs' secretome and whole cell lysate.
The proteomic phenotyping of human and ovine PBMCs revealed 32 orthologous CD antigens with no significant difference in abundance levels between species.The surface markers indicated the presence of six distinct immune cell subsets in both human and ovine PBMCs, CD14 + CD16 + monocytes, CD3 + CD4 + T cells, CD3 + CD8 + T cells, CD19 + CD22 + B cells, CD11c + mDCs, and CD244 + CD352 + NK cells, consistent with established classifications 63,[126][127][128] .However, ovine PMBCs also contained a unique WC1 + γδ T cell subset, not detected in hPBMCs.While the comparable abundance levels of immune cell subset markers indicate a similar composition of ovine and human PBMCs, establishing a crucial foundation for modeling inflammatory responses and interpreting subsequent proteomic shifts in both species, the presence of a unique T-cell subset introduces a potential confounding species-specific difference.
Comparative proteomic profiling of hPBMCs and oPBMCs revealed an overlap of approximately half (47.8%, 2790 proteins) of the entire cell lysate proteome, and one-third (32.8%, 988 proteins) of the secretome proteome between the two species.However, upon inflammatory stimulation, only seven differentially abundant proteins (IL1B, IFIH1, CCL4, ISG20, IL1RN, APOBEC3A, and PDCD11) were shared between sheep and humans, while 169 were specific to humans and 64 species-specific to sheep.This limited overlap, although consistent with comparable studies exploring proteome/transcriptome changes in human and mouse during Th17 cell differentiation 129,130 , is even more pronounced at the protein level of PBMCs in the current study.Two primary factors may contribute to this lack of overlap.First, considerable heterogeneity and compositional variations exist among circulating PBMCs in different species 50,131,132 .For instance, γδ T cells, a subset of lymphoid cells, typically constitute 0.5-10% of circulating T lymphocytes in adult humans 132,133 , while in adult sheep they represent up to 17% 131,133 and in lambs 30-60% [134][135][136][137] .This heterogeneity may explain the successful identification of CD markers specific to the WC1 + γδ T cell subset in oPBMCs, a subset not detected in our hPBMCs samples.Second, inter-species differences in the cellular machinery arise from the intricate interplay between the  87,138 .Therefore, incorporating signals not only at the level of orthologous individual molecules (proteins) but also within functional sets, complexes, and pathways is crucial when translating findings from ovine immunology to the human setting.
Using PPI networks and GO analyses, we identified 16 shared GO terms between both species with a strong representation of inflammatory-related processes.Enrichment analysis identified the major shared biological process "immune and inflammatory responses", encompassing high-enrichment terms such as "leukocyte activation", "leukocyte migration", "leukocyte degranulation", "leukocyte-mediated immunity", "adaptive immune response", "innate immune response", and "cytokine production involved in immune response" 139 that are associated with well-established consequences of inflammatory activation of PBMCs by LPS/PHA 55,107,140 .
Network analysis revealed five potential hub proteins in sheep and humans, primarily associated with inflammatory processes 55,98,101,141,142 .In sheep, the hub proteins included STAT1, IL1B, IRF4, STAT3, and IL17A, while in humans, they comprised CXCL10, CXCL8, IL1B, IL6, and TNF.Notably, IL1B, a potent pro-inflammatory cytokine with a pivotal role in orchestrating innate and adaptive immune responses 143,144 , emerged as the sole hub protein shared between both species, detected in the whole cell lysates and secretomes of PBMCs.
Considering that the secretome samples and the cell lysate samples were collected simultaneously, the obtained secretomes contain accumulated proteins synthesized and secreted over the incubation time (6 h in the current study), whereas the proteins obtained from the cell lysates give insight in the current cell status at the time point of collection.The current study utilized integrative global mass spectrometry-based proteomics analyses of both the secretome (extracellular) and whole cell lysate (intracellular) of PBMCs to assess of the pattern of activation/ inhibition in shared signaling pathways and their underlying molecular mechanisms across both species and gain insight into the intricate regulatory mechanisms.In response to inflammatory stimulation, 17 canonical pathways, associated with the DAP of PBMCs of both species, exhibited consistent trends of activation/inhibition in both the secretome and the cell lysates (e.g., interferon signaling, inflammasome pathway, Pathogen Induced Cytokine Storm Signaling Pathway, acute phase response signaling, ERK/MAPK Signaling, CXCR4 Signaling, NF-κB Activation by Viruses, IL-33 Signaling, IL-8 Signaling, Integrin Signaling, etc.), emphasizing a high degree of conservation in immune and inflammatory responses across species.This observed conservation can be attributed to the substantial evolutionary conservation of inflammatory signaling and its transcriptional mechanisms in vertebrates 145,146 , despite variations in susceptibility and physiological differences between species [147][148][149][150] .For instance, the substantial homology between ovine and human Toll-like receptors (82-88% homology) 151,152 , as well as the close similarity in genomic responses and cardiopulmonary hemodynamics of sheep and humans challenged with lipopolysaccharide (LPS), further support the conservation 70,[152][153][154][155][156][157][158] .
However, 7 divergent canonical pathways exhibited different trends of activation/inhibition in humans and sheep highlighting potential species-specific adaptive differences in the regulation of intracellular signaling pathways.Specifically, initial activation of "IL-6 signaling", "HMGB1 signaling", "p38 MAPK signaling", "S100 family signaling pathway", "IL-17 signaling", "Mitochondrial Dysfunction", and "Glycolysis I" was evident in the secretome of both species but rapid inhibition only in the whole cell lysate of sheep.These finding align with previous studies suggesting that differences in chemokine and cytokine expression and the response of various cell types to inflammatory cytokines across species might be related to species variability in regulation of inflammatory signaling pathways 70,[159][160][161] .Inflammatory pathways are finely tuned by interconnected activating and inhibitory waves that delicately adjust the magnitude and duration of the inflammatory response over time to prevent tissue damage [162][163][164][165] .Thus, considering temporal changes in pathway regulation 163,[166][167][168] is crucial when translating pathways between sheep models and humans in future studies.
The lack of traditional immunochemical validation assays, primarily due to scarce sheep-specific antibodies, presents a methodological limitation of this study.However, Mass Spectrometry proteomics provides indirect validation by detecting proteomic patterns that are consistent with previously validated research 49,[51][52][53]140,[169][170][171][172][173] . Additionall, the MS-data provide a foundation for further refinement of the design of specific ovine antibodies for immuno-based analytical methods in future studies investigating immune repertoires in health and disease.
In conclusion, this cross-species comparative proteomics study sheds light on the intricate differences and shared aspects of the in vitro inflammatory response in ovine and human PBMCs, underscoring the importance of a judicious model selection to optimize the translatability of findings and uphold ethical standards in research.While significant similarities were found in conserved inflammatory pathways and biological processes, recognizing and addressing inherent species-specific differences is imperative when interpreting results of inflammation research results conducted in the ovine model.For inflammatory processes exhibiting divergence between the two species, the utilization of human-derived in vitro models or alternative animal models is recommended to optimize translational potential.Evidence-based selection of fit-for-purpose models ensures scientific quality and relevance of pre-clinical inflammation research while minimizing unnecessary animal use.

Figure 1 .
Figure 1.Optimization of the ovine peripheral blood mononuclear cell (oPBMC) isolation protocol.(a) Optimization of the blood dilution, density gradient and centrifugation parameters based on (b) PBMC separation quality.Selection of the density gradient based on (c) PBMC yield and (d) PBMC purity and composition.(e) Mass Spectrometry assessment of purity based on granulocyte-specific cluster of differentiation (CD) antigens and specific proteins associated with platelets and plasma.(f) Mass Spectrometry-based identification of orthologous CD antigens, indicating the presence of seven immune cell subsets.

Figure 3 .
Figure 3. Comparative protein-protein interaction (PPI) networks and functional enrichment in ovine and human PBMCs, showing (a) the ovine main PPI cluster, (b) the human main PPI cluster, (c) functional enrichment of biological processes in ovine differentially abundant proteins (DAPs) and (d) functional enrichment of biological processes in human DAPs. °C,

Table 2 .
Comparison of CD marker expression between ovine and human control and activated PBMCs (*indicates p < 0.05).

Table 3 .
Comparison of surface marker ratios across species and activation states.

Table 5 .
Comparative analysis of canonical pathways in human and ovine PBMCs based on differential proteomic expression across secretome (HSE human PBMC secretome, SSE ovine PBMC secretome) and whole cell lysate (HCL human PBMC whole cell lysate, SCL ovine PBMC whole cell lysate) datasets.